hydroxyapatite nanofibrous scaffold, in bone tissue engineering

Applied Materials Today 5 (2016) 52–67

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Investigating the potential of combined growth factors delivery, from non-mulberry silk fibroin grafted poly(␧-caprolactone)/hydroxyapatite nanofibrous scaffold, in bone tissue engineering Promita Bhattacharjee a,∗ , Deboki Naskar b , Tapas K. Maiti b , Debasis Bhattacharya a , Subhas C. Kundu b,c a

Materials Science Centre, Indian Institute of Technology Kharagpur, West Bengal 721302, India Department of Biotechnology, Indian Institute of Technology Kharagpur, West Bengal 721302, India c Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, South Korea b

a r t i c l e

i n f o

Article history: Received 8 May 2016 Received in revised form 22 July 2016 Accepted 10 September 2016 Keywords: Non-mulberry silk fibroin Aminolysis Growth factors Alternative soaking Bone tissue engineering

a b s t r a c t Mineralized scaffolds have the advantage of better mimicking the natural bone structure and thus show an improved potential for bone tissue engineering. This study uses cycles of alternative soaking to deposit hydroxyapatite (HAp) layers upon non-mulberry silk fibroin (from Antheraea mylitta) grafted poly(␧caprolactone) nanofibrous matrices. Alternate soaking, of one through three cycles, is used due to its simplicity and deposition efficiency. HAp deposition improved mechanical strength of the scaffolds up to two cycles of soaking (by nearly 75%). Analysis of mechanical properties, bioactivity and in vitro study results (with MG-63 cell line) showed scaffolds fabricated using two-cycle soaking to be the most suitable. These constructs were loaded with growth factors (transforming growth factor beta (TGF-␤) 4 ng and bone morphogenic protein-2 (rhBMP-2) 100 ng) using carbodiimide-coupling reaction. The following three different combinations of growth factor loaded composites were analyzed: solely rhBMP-2, solely TGF-␤ and rhBMP-2-TGF-␤ combinations. Scaffolds with both growth factors supported cellular activity and proliferation better (p < 0.01), generated greater calcium deposits (p < 0.01), facilitated early cell differentiation and yielded significantly better expression of genes linked to bone growth (p < 0.05). These dual growth factor loaded scaffolds are mechanically robust and enhance cell proliferation and early differentiation of osteoblast-like cells. They thus show potential of being further optimized for use in bone tissue engineering. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Natural bone’s multi-scale, ordered structure combines organic polymers with inorganic crystals [1,2]. Designing bone tissue engineering scaffolds based on this multi-scale, organic/inorganic composite structure is advantageous [3]. Hydroxyapatite (HAp), due to its close resemblance to bone tissue’s inorganic phase, has become the most investigated option for the inorganic component [4]. As cost and complexities in purification limit use of collagen for the organic component [5], the following synthetic and biopolymers have been explored for suitable alternatives: poly(l-lactic acid) [6], poly(␧-caprolactone) [7], gelatin [8] and silk fibroin (SF) [9]. Silk’s natural strength, biocompatibility, biodegradability, water and oxygen permeability and nominal immune reactivity make

∗ Corresponding author. E-mail address: [email protected] (P. Bhattacharjee). http://dx.doi.org/10.1016/j.apmt.2016.09.007 2352-9407/© 2016 Elsevier Ltd. All rights reserved.

it an evident option for bone tissue engineering [10]. This work uses silk fibroin from Antheraea mylitta (NSF) – the Indian tropical tasar silkworm. Tasar silk is known to enhance osseointegration in vitro [11], at least partly due to its inherent tri-peptide (Arg-Gly-Asp) integrin-binding RGD sequences promoting cell adhesion and subsequent proliferation [12]. Previously, biocompatible, biodegradable, electrospun PCL nanofibrous matrices, grafted with NSF (Scheme 1), and imitating bone ECM, were created [13]. The next logical step, their mineralization, was carried out for the current work. SF-HAp composites have been fabricated using co-precipitation and thermo-compression [14,15]. Electrospun matrices’ high porosity makes them suitable for mineralization [16]. Due to ionic interactions, “alternate soaking” method can provide substantial HAp deposits expeditiously [17]. Multiple growth factors (various isoforms of TGF-␤s and BMPs) contribute to natural bone’s development and regeneration [18–21]. Growth factor uses have shown results ranging from beneficial to detrimental [22,23]. Studies indicate requiring supraphysiological amounts of BMPs to obtain benefits in clinical usage.

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Scheme 1. The schematic representation of non-mulberry silk fibroin grafting via aminolysis on PCL nanofibrous matrix.

Requirement of such amounts makes the process uneconomical [22] and raises potential for adverse effects like tissue swelling, radiculopathy, seroma and bone formation heterotopy [24]. As signalling routes of BMP-2 and TGF-␤ have cross-interactions [25], our starting hypothesis is that with suitable scaffold characteristics, physiological amounts of growth factors can be delivered, in appropriate combination, for enhancing bone regeneration potential. The alternative soaking method couples HAp to NSF, without using heat, though graft polymerization with free radical initiation, aided by 4-methacryloyloxyethyl trimellitate anhydride (4-META, Scheme 2). 4-META has been successfully and safely used in clinical dentistry [26]. Similar surface modification can be followed by electrodeposition for mineralization [27]. Alternative soaking, unlike electrodeposition, does not require specialized set-up and precious metal electrodes (commonly, platinum). Hence, the applicability of this simple and straightforward process in fabricating nanocomposite scaffolds, that may support and sustain osteogenic cell growth, needs investigation.

Three nanofibrous scaffolds were fabricated using respectively 1, 2, and 3 coating cycles and compared on the basis of their mechano-physical characteristics, bioactivity and support of osteoblast-like cells, in vitro. Depending on the result of bioactivity and mechanical property, HAp deposited by 2-cycle coating on silk fibroin grafted PCL nanofibrous scaffold was used for subsequent detailed in vitro studies, after incorporating different combinations of the growth factors, as per our starting hypothesis. 2. Materials and methodology 2.1. Materials Major materials used were the following: poly(␧-caprolactone) (PCL, Mol. wt. = 80,000), chloroform, glutaraldehyde, calcium chloride (CaCl2 ), disodium hydrogen phosphate (Na2 HPO4 ), pentaethylene glycol dodecyl ether (surfactant), l-glutamic acid, thiazolyl blue tetrazolium bromide (MTT) (Sigma, St. Louis, USA),

Scheme 2. The schematic representation of graft polymerization with 4-META onto the silk fibroin by free radical initiation and synthesis of NSF-PCL/HAp composite by ionic interaction.

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Table 1 Composition details, surface roughness (Rq), tensile properties and dynamic contact angle, with goniometer image (n = 3, mean ± SD), of the non-mulberry silk fibroin grafted PCL nanofibrous matrix (NSF-PCL), and HAp-deposited NSF-PCL by 1 cycle (NSF-PCL/1C), 2 cycles (NSF-PCL/2C) and 3 cycles (NSF-PCL/3C) of alternative soaking method. Compositions

Compositions details

Surface roughness (Rq) (nm)

Ultimate tensile strength (MPa)

Elongation at break (%)

Advancing contact angle (◦ )

Receding contact angle (◦ )

Mean contact angle (◦ ) with Goniometer image

NSF-PCL

Non-mulberry silk fibroin grafted PCL nanofibrous matrix (without HAp coating)

293.23 ± 0.21

10.23 ± 0.75

57.13 ± 3.52

36.89

32.61

34.75 ± 2.57

NSF-PCL/1C

Deposition of HAp on non-mulberry silk fibroin grafted PCL nanofibrous matrix by alternative soaking cycle (1 cycle)

320 ± 0.06

12.01± 0.34

82.57 ± 5.62

27.14

19

23.07 ± 1.76

NSF-PCL/2C

Deposition of HAp on non-mulberry silk fibroin grafted PCL nanofibrous matrix by alternative soaking cycle (2 cycles)

396.53 ± 0.11

17.98 ± 0.82

120.34 ± 5.91

18.98

16.06

17.52 ± 1.57

NSF-PCL/3C

Deposition of HAp on non-mulberry silk fibroin grafted PCL nanofibrous matrix by alternative soaking cycle (3 cycles)

429.31 ± 0.26

14.52± 0.55

115.32 ± 4.17

16.73

13.91

15.32 ± 2.16

1,6-hexanediamine (TCI, Japan), 4-methacryloyloxyethyl trimellitate anhydride (4-META) monomer (Polysciences, Inc., USA), sodium dodecyl sulfate (Mol. Wt. = 288.38) (J.T. Baker, NJ, USA), polyethylene glycol (Mol. Wt. = 6000), ammonium peroxodisulfate (APS, initiator), potassium hydroxide (KOH, Merck, India, ionizing reagent), cellulose dialysis tubing with cut-off 12,000 and 3500 kDa (Pierce, USA), tissue culture grade polystyrene plastic flasks and plates (Tarsons, India), Dulbecco’s modified eagle medium (DMEM), foetal calf serum, trypsin, EDTA, penicillin–streptomycin antibiotics (Gibco BRL, USA) and alamar blue (Invitrogen, USA). Antheraea mylitta silkworms were reared at IIT Kharagpur’s silk farm till their late fifth instar, when they were about to start spinning. Human osteoblast-like cells (MG 63) were procured from the National Centre for Cell Science (NCCS), Pune, India. 2.2. Isolation of silk fibroin from silk glands and immobilization on PCL nanofibrous matrix via aminolysis Silkworm larvae in their late fifth instar were dissected to obtain the silk glands, by following the procedure detailed in previous works [12]. Fibroin was dissolved in a room temperature aqueous solution of 5 mM EDTA, 10 mM Tris (pH 8.0) and 1%w/v sodium dodecyl sulfate. The solution was dialyzed, removing surfactant traces and adjusting fibroin concentration at 2 wt%. Methodology detailed in our previous work was used to electrospin nanofibrous PCL scaffolds and graft NSF onto them, via aminolysis [13]. Grafting schematics have been presented in Scheme 1. 2.3. Graft polymerization with 4-META onto NSF-PCL, and NSF-PCL/HAp composite synthesis by alternate soaking method A free radical initiation technique, aided by 4-META, was used to graft HAp onto the aforementioned nanofibrous matrices. Details of this procedure have been expounded in [26,28]. Following polymerization, matrices were washed with acetone and distilled

water, to remove any homopolymers or unreacted monomers, and vacuum dried for 24 h. Matrix weight gain was calculated using Eq. (1). Weight gain (wt %) =

Wf − Wi Wi

× 100(%)

(1)

Here, Wi is NSF-PCL matrices’ initial weight and Wf is the final weight of these matrices following poly(4-META) grafting and drying. Soaking the grafted matrices in 0.01 M aqueous KOH opened the 5-member ring of 4-META. Subsequently, HAp deposition was carried out using alternate soaking method. Matrices were put in a petri dish set on a mechanical shaker – 150 rpm, at 37 ◦ C – with 200 mM CaCl2 , for 1 h. Post an hour of immersion, matrices were taken out, blotted with filter paper to remove excess moisture and submerged in 120 mM Na2 HPO4 solution, with the same set-up, for an hour. This combination was termed as one cycle. One (NSFPCL/1C), two (NSF-PCL/2C) and three (NSF-PCL/3C) cycle deposits were carried out. Multi-layered nature of the HAp deposition was confirmed by measuring thickness of scaffolds after each cycle of deposition, using a stylus profilometer (Veeco Dektak 3). Scaffold thickness consistently increased after each cycle. These matrices with HAp coatings were washed with distilled water and dried for 24 h in air at room temperature. Their composition is presented in Table 1. Schematic representation of the reaction and the procedure is given in Scheme 2. 2.4. Physical characterizations of the composite nanofibrous scaffolds: The nanofibrous scaffolds were observed under analytical SEM (A-SEM, ZEISS EVO 60 Scanning Electron Microscope, Carl ZEISS SMT, Germany) to examine their morphology and elemental composition (through EDAX). SEM images were analyzed using Image J® (release 1.47 for Windows) to determine mean

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Table 2 Type of growth factors loaded in different proportions on HAp deposited (2 cycles) non-mulberry silk fibroin grafted PCL nanofibrous scaffolds (NSF-PCL/2C), with proper abbreviations. Compositions

Compositions details

Growth factor

Loading efficiency of growth factor (ng)

NSF-PCL/2C

Deposition of HAp on non-mulberry silk fibroin grafted PCL nanofibrous matrix by alternative soaking cycle (2 cycles) TGF-␤ loaded HAp-deposited non-mulberry silk fibroin grafted PCL nanofibrous matrix (2 cycles) rhBMP-2 loaded HAp-deposited non-mulberry silk fibroin grafted PCL nanofibrous matrix (2 cycles) TGF-␤ and rhBMP-2 loaded HAp-deposited non-mulberry silk fibroin grafted PCL nanofibrous matrix (2 cycles)

No growth factor

–

TGF-␤

4

rhBMP-2

100

TGF-␤ + rhBMP-2

(4 ng TGF-␤ + 100 ng rhBMP-2) = 104

T2C B2C T/B 2C

nanofiber diameter. Crystal (X-ray diffractometer, PW1710, Philips, Netherlands), chemical (FTIR, Nexus-870, Thermo Nicolet Corporation, USA) and thermal analyses (TGA, Perkin Elmer Pyris Diamond TG-DTA thermo-gravimetric analyzer) of the scaffolds were also carried out. The deposits formed were confirmed as being HAp through XRD (10–70◦ scanning range in 2, 2◦ /min speed), FTIR and TGA (room temperature to 800 ◦ C, in synthetic air (N2 :O2 = 80:20), at 10 ◦ C/min) analyses. Tensile tests on the scaffolds were conducted using a universal testing machine (UTM; Instron Electroplus, E1000), in a 25 ◦ C and 50% relative humidity environment (ASTM 638–5 standard, 5 kg load-cell, 3 mm/min extension rate). HAp deposition amount (wt%) and density (per unit scaffold volume) were calculated post each soaking cycle (n = 5), using Eqs. (2) and (3) respectively. Scaffolds had dimension of 1 × 1 × 0.1 cm3 . HAp deposition (w/w, %) =

w2 − w1 × 100 (%) W1

HAp formation density (mg/cm3 ) =

w2 − w1 1 cm × 1 cm × 0.1 cm

(2)

 mg  cm3 (3)

W1 is the poly(4-META) modified NSF-PCL scaffold’s weight before and W2 its weight after each soaking cycle. A goniometer (Data Physics Instruments, Filderstadt, Germany) measured hydrophilicity of the composite scaffolds. Topographic images from atomic force microscopy (AFM; Model 5100, Agilent Technologies, USA) were used to analyze surface roughness of 10 × 10 ␮m2 samples. Water uptake capacity of scaffolds was calculated using Eq. (4) (dry weight, Wdry and wet weight, Wwet ). Water uptake (%) =

Wwet − Wdry Wdry

× 100 (%)

cell adhesion, for the first hour after seeding, the scaffolds were kept in a humid ambient, at 37 ◦ C and 5% CO2 . These in vitro studies were carried over 14 days, while replacing the medium every alternate day. Cell viability (MTT) and proliferation (alamar blue) assay were performed at specific time points. Data from the preliminary set of in vitro studies and tensile testing showed the NSF-PCL/2C nanofibrous scaffold as the most promising choice. Hence, NSF-PCL/2C was used for growth factors incorporation and the detailed in vitro studies that followed. 2.6. Preparation of growth factors loaded composite scaffolds and release kinetics study of growth factors Growth factors, TGF-␤ (Sigma, St. Louis, USA) and rhBMP-2 (Sigma, St. Louis, USA), were covalently coupled onto NSF-PCL/2C through a carbodiimide-coupling reaction, with l-glutamic acid modification [29]. NSF-PCL/2C scaffolds were incubated for 6 h in an aqueous carbodiimide solution, at room temperature, with gentle stirring. This incubation triggered free terminal COOH groups on HAp-g surface. These scaffolds were rinsed before being placed in a shaker for 6 h, with 100 ng rhBMP-2, 4 ng TGF-␤ and 104 ng rhBMP-2 (100 ng) + TGF-␤ (4 ng) dissolved in coupling buffer. After 6 h, the scaffolds were washed with distilled water, to remove any impurities, and freeze dried. The nature of growth factor loaded scaffolds is summarized in Table 2, with relevant nomenclature. Growth factor inclusion process is presented in Scheme 3. Growth factor loaded scaffolds (100 ng dosage for rhBMP-2 and 4 ng for TGF-␤) were incubated in PBS and maintained at 37 ◦ C, over 28 days, to determine controlled release kinetics. Media were collected at specific day points and growth factor release was measured using the respective ELISA kits (Invitrogen, USA).

(4)

Ion concentration (Ca2+ ) in water, after soaking the composite scaffolds, was measured using Inductive Coupled Plasma spectrometry (ICP). Enzymatic degradation of scaffolds was carried out using 2 ␮g/ml concentration solution of proteinase K (Tritirachium album origin; Sigma–Aldrich, USA) in phosphate buffer solution and the degradation levels measured at day points 1, 7, 14 and 21. 2.5. Cell culture on the composite nanofibrous scaffolds The human osteoblast-like cells were cultured in a medium made of DMEM and 10% foetal calf serum, with 1% penicillin/streptomycin, till reaching 90% confluence. Subsequently, cells were trypsinized and centrifuged, before being resuspended in the media for counting. For cell seeding, scaffolds (1 × 1 cm2 ) were sterilized under UV light and with 70% ethanol, over 30 min. Scaffolds were washed in sterile PBS (pH 7.4) and then treated over 4 h with DMEM medium, to make them conducive for the cells. They were partially dried, for 2 h before actual seeding, to allow better penetration of cells. Cell suspension medium (15 ␮l) containing 8 × 104 cells were added drop-wise upon the nanofibrous scaffolds. To boost initial

2.7. Cell culture and in vitro biocompatibility studies Cell line maintenance and pre-seeding scaffold was as described in Section 2.5. Cell suspension (15 ␮l) with 2 × 104 cells was added onto each nanofibrous scaffold. In vitro culture duration was 14 days and the medium was replaced on every alternate day. Cellular adhesion [13], viability (MTT), proliferation (alamar blue) and total protein content (BCA assay kit, Thermo Fisher Scientific) assessment on the cell-laden scaffolds were performed at specific time points. 2.7.1. Gene expression by real-time RT-PCR After 14 days of culture, for total RNA extraction, cell-laden, growth factor loaded scaffolds were transferred into 2 ml plastic tubes containing 1.5 ml Trizol solution (Invitrogen, USA). The scaffolds were incubated for 15 min and then centrifuged for 10 min, at 12,000 g and 4 ◦ C. Supernatant was collected into fresh tubes, with 200 ml chloroform, and incubated at room temperature, for 5 min. This was followed by 15 s of gentle stirring and 15 min of further centrifugation at 12,000 g and 4 ◦ C. Resulting supernatant was placed into RNeasy Plus mini-spin column (Qiagen, Germany). Manufacturer’s protocol was adhered to for washing and

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Scheme 3. The schematic representation of growth factor/(s) incorporation on NSF-PCL/HAp composite by carbodiimide-coupling reaction.

RNA elution. High-capacity cDNA reverse transcription kit (Applied Biosystems, USA) was employed for RNA to cDNA reverse transcribing. Real-time PCR (RT-PCR) was carried out with SYBR Green (Applied Biosystems, USA), in the ABI Prism® 7000 Sequence Detection System (Applied Biosystems, USA). Conditions for the RT-PCR were optimized before engaging in the actual runs. A reaction volume of 50 ␮l was fixed upon. This volume contained 5 pmol/ml forward and reverse primers, 5 ␮l cDNA template and the SYBR Green supermix. RT loading platform was used for plate loading. The cycling steps consisted of a denaturation step (8 min 45 s, 95 ◦ C), followed by 45 cycles, with each cycle having 30 s at 95 ◦ C, 58 ◦ C and 72 ◦ C, sequentially. During each cycle, data were collected at the 72 ◦ C phase. Cycle threshold (CT) values were calculated using Applied Biosystems’ Relative Quantification software. High-purity gene-specific primers for relevant bone-specific genes (alkaline phosphatase (ALP), osteopontin (OPN), osteonectin (OST), collagen I, osteocalcin (OCN), Runx2 and bone sialoprotein (BSP)) and the housekeeping gene GAPDH had been designed following previous reports (Table 3) [30–32]. These were synthesized commercially (MWG-Biotech AG Ltd, India). Each target gene’s relative expression levels were normalized against the housekeeping gene’s Ct value (2−Ct formula, Perkin Elmer User Bulletin, s = / 2).

2.7.2. Live/dead assay, cytoskeleton organization and cell morphology Qualitative measurement of cellular viability (Live/dead assay kit, Molecular Probes, USA) was performed after 5 days of culture, using confocal microscope (Olympus FV 1000, Olympus, Japan). Live/dead assay procedure used manufacturer’s protocol (Molecular Probes, USA). A dye solution was formed using 40 nM calcein AM and 20 nM ethidiumhomodimer added to DMEM, without FBS. This showed green live cells (due to calcein staining) and red dead

cells (due to ethidiumhomodimer staining) under confocal examination. Imaging from confocal microscope and SEM were also used to investigate cell morphology and distribution. Cell-laden scaffolds were fixed by 1 h treatment with 4% paraformaldehyde. For examination under confocal microscope, fixed cell-laden scaffolds were treated with 0.1% Triton X-100 solution in BSA, for 5 min, to permeabilize the cells. They were subsequently blocked using 1% bovine serum albumin (BSA), over an hour. Alexa Fluor® 488 and Hoechst 33342 were used to stain actin filaments and nuclei, respectively. Olympus FV 1000 Advanced software (version 4.1, Olympus, Japan) was used for post-processing the confocal images. Samples of fixed, cell-laden scaffolds, to be examined under SEM, were dehydrated by placing them for 20 min each in 6 ethanol–water gradients (increasing from 50 to 100% v/v ethanol, at 10% v/v steps). Finally, they were briefly exposed to isoamyl acetate and vacuum dried. These constructs were sputter coated with gold and examined at 15 kV under FESEM (FESEM, SUPRA 40).

2.7.3. Alkaline phosphatase (ALP), Alizarin Red-S and calcium mineralization assay Kim et al.’s [33] protocol was used for spectrophotometric determination of ALP production, an early osteogenic marker, from the cultured MG-63 cells. ALP activity has been reported by normalizing the values with respect to incubation duration and cell count, i.e. ␮mol/min/104 cells. Cell counts were obtained via DNA analysis (Genomic DNA Purification kit Thermo Fisher Scientific). As Alizarin Red-S (ARS) has the ability to selectively bind with calcium salts, ARS (40 mM ARS) staining, followed by use of 10% cetylpyridinium chloride, was employed to detect and quantify mineralization due to cell growth. Quantification of calcium deposits on the cell-scaffold constructs was done using cresolphthalein complexone (Sigma), as reported by Kim et al. [33].

Table 3 RT-PCR primer sequences, forward and reverse, used in the current gene expression study. Genes Runx2 OCN Osteonectin OPN ALP BSP COL I GAPDH

Forward primer 

Reverse primer 

5 -GCTTCTCCAACCCACGAATG-3 5 -AAAGCCCAGCGACTCT-3 5 -ACAAGCTCCACCTGGACTACA-3 5 -GACGGCCGAGGTGATAGCTT-3 5 -TCAGAAGCTCAACACCAACG -3 5 -CAGGGAGGCAGTGACTCTTC-3 5 -TCCTGCCGATGTCGCTATC-3 5 -AGGTCGGTGTGAACGGATTTG-3



Reference 

5 -GAACTGATAGGACGCTGACGA-3 5 -CTAAACGGTGGTGCCATAGAT-3 5 -TCTTCTTCACACGCAGTTT-3 5 -CATGGCTGGTCTTCCCGTTGC-3 5 -TTGTACGTCTTGGAGAGGGC -3 5 -AGTGTGGAAAGTGTGGCGTT-3 5 -CAAGTTCCGGTGTGACTCGTG-3 5 -TGTAGACCATGTAGTTGAGGTCA-3

[30] [30] [30] [30] [31] [32] [30] [32]

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2.8. Statistical analysis Unless otherwise specified, data are given as mean ± standard deviation (SD) and sample size (n) was 3. One-way ANOVA, complemented with Tukey’s HSD test, was used to compare performance of different scaffold compositions. Following categorization and symbols were used for observed significant differences: *** p < 0.001; ** p < 0.01; * p < 0.05. Statistical analysis was performed in the R statistical environment. 3. Results and discussion 3.1. Weight gain of matrix after graft polymerization Scaffold weight gains, after polymerization with poly (4-META), plotted with respect to reaction time have been presented in supplementary data (Fig. S1). Weight gain increased with reaction time, eventually reaching a near-stable value of ∼18.93 wt%, with good repeatability. This suggested low graft efficiency. A peak value is

57

quickly reached, likely due to the steric hindrance between side chains of the NSF substrate and 4-META [26]. All results discussed in this work were obtained using these scaffolds, with 18.93% weight gain. 3.2. Physical characterizations of the composite nanofibrous scaffolds 3.2.1. Increase of scaffold thickness after deposition of HAp Scaffold thickness increased with every HAp deposition cycle, as shown in supplementary figure, Fig.S2. This increase was assumed to be caused due to multi-layered HAp deposition on the matrix. The deposit was confirmed as being HAp through further analysis using XRD, FTIR and EDAX. 3.2.2. Morphology analysis SEM images of the electrospun NSF-PCL matrices displayed bead-less, uniform, and smooth nanofibers, with 452 ± 16 nm average fibre diameter (Fig. 1(a)). These matrices had random,

Fig. 1. Scanning electron micrographs and corresponding EDAX analysis of the different HAp deposited composite nanofibrous scaffolds (NSF-PCL, NSF-PCL/1C, NSF-PCL/2C and NSF-PCL/3C) fabricated by alternative soaking method. For all substrates, Ca/P was about 1.6, which is almost equal to biological hydroxyapatite (Ca/P = 1.67).

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Fig. 2. (a) X-ray diffraction patterns of NSF-PCL, NSF-PCL/1C, NSF-PCL/2C, and NSF-PCL/3C composite nanofibrous matrices indicated that after deposition of HAp, the crystalline characteristics of the constitutive components – HAp, PCL and NSF – still existed. (b) ATR-FTIR spectra of NSF-PCL, NSF-PCL/1C, NSF-PCL/2C, and NSF-PCL/3C composite nanofibrous matrices. The HAp-deposited composite nanofibrous matrices reveal peaks of PCL, NSF, and HAp without any major alterations.

non-woven architecture, with interconnected pores, thus, mimicking natural bone’s ECM. Cycles of alternative soaking successfully mineralized these matrices with HAp. As may be observed from the SEM images, a single cycle (NSF-PCL/1C) gave the matrices a partial coating of HAp (Fig. 1(b)). Two complete cycles (NSF-PCL/2C) covered almost the entire surface with HAp (Fig. 1(c)), while three cycles (NSF-PCL/3C) lead to bulk HAp formation (Fig. 1(d)). EDS spectra confirmed the deposited material to be calcium phosphate (Fig. 1). EDS analysis showed a Ca/P ratio of 1.76 after 3rd cycle, i.e., slightly higher than the value of 1.67, from stoichiometry of HAp’s chemical structure Ca10 (PO4 )6 (OH)2 . This implies the HAp formed was calcium rich. With 2 cycles of coating, Ca/P was around 1.67. This value agreed well with the stoichiometric value. With increase in number of soaking cycles, the carbon and nitrogen contents decreased while Ca and P contents increased. As evidenced from SEM and EDS, the scaffolds were mineralized with HAp and the alternate soaking method gave near-uniform deposits. The method is a tried and tested method and relies on the strong affinity between HAp and 4-META prompting an ionic interaction [26] (Scheme 2). a-plane of HAp has Ca2+ ions on its surface [26]. The fine fibrous structure of NSF-PCL matrix (∼452 nm), consisting of sub-micron-sized fibres, presents a large surface area to volume ratio and a structure with interconnected porosity. Both factors have a critical role to play in transporting nutrients and oxygen to the cells and are hence quite essential for cell growth in vitro and in vivo. 3.2.3. Crystal and chemical structure and thermogravimetric analysis Crystallinity of the HAp deposits was analyzed using XRD (Fig. 2(a)). NSF-PCL matrices showed two intense and sharp peaks ascribable to PCL, at 2 = 21.5◦ and 23.8◦ (Fig. 2(a)). These may

be assigned to PCL’s (1 1 0) and (2 0 0) planes [34]. NSF-specific crystalline peak was observed at 2 = 17◦ , confirming the ␤-sheet structure resulting from ethanol treatment [34]. Characteristic crystalline peaks of the components in XRD analysis showed that HAp-deposited samples retained crystalline characteristics of HAp, PCL and NSF. Peaks corresponding to PCL were weakened. Peaks corresponding to HAp crystalline structure were observed at 2 = 26◦ , 31.8◦ [36]. As the number of soaking cycles increased, intensity and sharpness of HAp peaks improved. This implies that HAp crystallinity depended on the number of soaking cycles. ATR-FTIR spectroscopy of mineralized nanofibrous scaffolds is presented in Fig. 2(b). Of the NSF-PCL nanofibrous matrix’s major vibration peaks, the following strongest bands can be associated to PCL: 1726 cm−1 for C O and C–O groups and peaks at 1367, 2864 and 2940 cm−1 for the C–H bond in PCL [37]. For NSF, these vibration peaks ascribable to amide groups were observed (Fig. 2(b)): 1650–1630 cm−1 for amide I (C O stretching), 1540–1520 cm− 1 for amide-II (secondary NH bending, ␤-sheet structure) and 1270–1230 cm−1 for amide III (C–N and N–H) [35]. Absorption peaks corresponding to ∼1535 cm−1 and ∼1648 cm−1 point to ␤sheet structure (amide II). Additional characteristic peaks observed were the following: ∼1023 cm−1 for P–O stretching of phosphate group, ∼560 and 600 cm−1 for P–O bending and ∼3384 cm−1 for O–H stretching [38]. Intensity and broadness (especially O–H stretching) of the absorbance bands was boosted with increasing number of soaking cycles. Data thus obtained further HAp formation on the matrices. During TGA analysis, weight loss onset was at 321 ◦ C for NSFPCL nanofibrous matrix (Fig. 3(a)), reaching 100% at 545 ◦ C. Onset temperature for weight loss of the scaffolds reduced as HAp concentration increased with number of soaking cycles. Following complete thermal degradation, the residual weight was used to calculate the scaffold’s HAp content. This yielded similar values

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Fig. 3. TGA analysis in term of (a) % of weight and (b) % of weight derivatives of NSF-PCL, NSF-PCL/1C, NSF-PCL/2C and NSF-PCL/3C composite nanofibrous matrices. (c) Percentage increase in deposition of HAp on the poly(4-META) modified NSF-PCL matrix was significant following each cycle (*** p < 0.001 and ** p < 0.01). (d) Effects of soaking cycle on the amount of HAp formed on poly(4-META) modified NSF-PCL matrix.

to those obtained from calculating weight of HAp deposited following each cycle of soaking. These were 11.42%, 21.5% and 32.7% for NSF-PCL/1C, NSF-PCL/2C and NSF-PCL/3C, respectively. The temperature for highest weight loss also reduced with HAp concentration (Fig. 3(b)): 392 ◦ C, 381 ◦ C, 369 ◦ C and 352 ◦ C for NSFPCL, NSF-PCL/1C, NSF-PCL/2C and NSF-PCL/3C, respectively, thus indicating reduced thermal stability of scaffold with rise in HAp content. 3.2.4. Change of weight of scaffolds and amount of HAp formation after soaking cycles HAp deposition weight on the NSF-PCL scaffolds increased directly as the number of soaking cycles (Fig. 3(c)). Weight rise was 11.2 ± 05% (w/w), 21.12 ± 03% (w/w) and 32.67 ± 07% (w/w) for one, two and three cycles, respectively. About 12.3 mg/cm3 , 28.9 mg/cm3 and 62.9 mg/cm3 of HAp formed after one, two and three cycles, respectively, and is presented in Fig. 3(d). 3.2.5. Wettability and surface topography of the scaffolds Scaffold hydrophilicity has an important effect on its biocompatibility. Contact angle measurements of the fabricated scaffolds (Table 1) showed reducing hydrophobicity of NSFPCL with increase in HAp content. At least partly the trend

is because of alternate soaking achieved near-uniform deposits of HAp on the matrix surface. This was similar to the results of Lee et al. [39], who found that HAp addition made the poly(lactide-co-glycolide) (PLGA) surface hydrophilic. Mineral nucleation occurred at sub-microscopic level on the surface and was aided by the presence of NSF, without adversely impacting wettability. Scaffold’s surface texture and properties are consequential for cell attachment and proliferation. AFM was used for characterizing surfaces of the fabricated scaffolds. The AFM images (Fig. 4 (a)–(d)) indicate clear changes to surface characteristics post mineralization. Composite scaffold’s surface displayed granular structures and spiny ridges. Efficient nucleation of HAp can be ascribed to presence of NSF in the scaffold. Processing the AFM images using Pico Image® software gave RMS surface roughness (Rq) of the scaffolds (Table 1). Rq was increased with number of deposition cycles while the NSF-PCL matrix had the minimum Rq (293.23 nm). These differences are statistically significant (p < 0.01). Enhanced surface roughness is likely to provide enhanced surface area for cellular adhesion and improved scope for serum and medium proteins, thus improving cell growth [13]. Nanometre range surface roughness can also enhance adhesion and proliferation of osteoblasts, as reported by Deligianni et al. [40] and Webster et al. [41].

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Fig. 4. The AFM pictographs of composite nanofibrous matrices (NSF-PCL, NSF-PCL/1C, NSF-PCL/2C and NSF-PCL/3C) show the surface topography (10 ␮m × 10 ␮m scan area). Surface roughness (Rq) was calculated from AFM images using Pico Image Basic Software and presented in Table 1.

3.2.6. Mechanical properties As seen from stress–strain curves for the composite scaffolds (Fig. 5(a)), the scaffolds adhered to Hooke’s law while strain <10%. Linear nature of the curves was lost as strain rose further. Mechanical properties of the scaffolds have been enlisted in Table 1. Addition of HAp significantly increased strain at break and tensile modulus of the scaffolds (p < 0.01). The NSF-PCL/2C matrix showed the best mechanical characteristics among all samples. Mechanical properties of NSF-PCL/3C were inferior to NSF-PCL/2C, even with higher HAp content. NSF-PCL/3C did possess superior mechanical properties compared to NSF-PCL/1C and NSF-PCL. HAp particles on a scaffold surface can act as mechanical reinforcement by providing an additional energy dissipation medium. Their mobile nature

allows them to reorient and realign during an imposed deformation, making temporary cross-linkages across polymeric chains. This leads to localized zones of high mechanical strength [42]. Increasing amount of HAp deposited on surface must adversely affect mobility of particles and reduce energy dissipation through realignments and linkage formation. Thus, NSF-PCL/2C could have better mechanical characteristics than NSF-PCL/3C. 3.2.7. Water uptake, calcium ion elution and enzymatic degradation studies Water uptake of composite scaffolds reduced with increase in number of soaking cycles (Supplementary data, Fig. S3(a)). It reached a stable value for NSF-PCL/2C and NSF-PCL/3C scaffolds in

Fig. 5. (a) Stress–strain curves of NSF-PCL, NSF-PCL/1C, NSF-PCL/2C and NSF-PCL/3C composite nanofibrous matrices. A substantial difference in tensile stress (MPa) was observed between different nanofibrous matrices. (b) Controlled release kinetics of rhBMP-2 and TGF-␤ from NSF-PCL/2C nanofibrous scaffold. A burst release over the first 3 days was observed for both growth factors. However, only 19.33% of the rhBMP-2 and 20.2% of the TGF-␤ were released from the composite nanofibrous scaffolds.

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3.5 h. This could have been because more HAp deposit meant higher water uptake and a stable value was reached in a finite amount of time. With just one cycle, HAp deposit was not enough to affect water uptake. These data signify that hydrophilicity, an important aspect of the scaffold for tissue engineering applications, can be modulated using mineralization of HAp on to the NSF-PCL scaffolds. Elution of calcium ions from the fabricated composite scaffolds, in ultrapure water, has been presented in Fig. S3(b) (supplementary data). The supernatant was analyzed using ICP measurements. Immersion immediately leads to leaching of calcium ion but the process plateaued soon. It is likely that calcium ions had stabilized on the silk fibroin’s ␤-sheet structure [43]. As the ion elution is not rapid, it may be concluded that the calcium ions bonded tightly to the ␤-sheet structure, through ionic interactions. Enzymatic treatment over 21 days lead to loss of 45% dry weight in NSF-PCL, 52% in NSF-PCL/1C, 58% in NSF-PCL/2C and 63% in NSFPCL/3C scaffolds (Supplementary data, Fig S4(a)). After day point 10, degradation rates slowed down. Compared to this, the fabricates incubated in PBS (pH 7.4), as control, did not show any appreciable

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weight loss (Supplementary data, Fig. S4(b)). This result was in line with the findings of Li et al. [44]. 3.3. Cell viability and proliferation on the composite nanofibrous scaffolds Up to day point 5, there was no difference in cell viability for the different composite scaffolds (supplementary data, Fig. S5(a)). Towards day 14, cell viability and proliferation both gradually improved. At day point 14, both viability (MTT assay) (Fig. S5(a)) and cell proliferation (alamar blue assay) (Fig. S5(b)) were highest for NSF-PCL/2C. Among the different NSF grafted PCL nanofibrous scaffolds with HAp deposition, NSF-PCL/2C was chosen for the subsequent steps, involving detailed in vitro studies following growth factor incorporation. This choice was based on the results of mechanical strength (Fig. 5(a)) and preliminary study of cell viability and proliferation (Supplementary data, Fig. S5). After 2-cycle coating of HAp, some porous structures on NSF-PCL/2C could be observed

Fig. 6. The response of osteoblast-like cells (MG-63) seeded on different growth factors loaded nanofibrous scaffolds and cultured for 14 days in 37 ◦ C and 5% CO2 , humidified atmosphere. (a) Initial cell attachment efficiency on different growth factors loaded scaffolds measured up to 6 h by counting the cells from suspension at each time point. NSF-PCL/2C (without growth factor) served as control. (b) Viability, (c) proliferation of cells and (d) the changes in content of the total protein of MG-63 cultured on different growth factors loaded nanofibrous scaffolds at various time points, indicating superior cell response on the dual growth factor loaded composite nanofibrous scaffolds (T/B2C), compared to single growth factor loaded scaffolds (T2C and B2C) and control (NSF-PCL/2C). *** p < 0.001, ** p < 0.01 and * p < 0.05, n = 3 at each time point (One-way ANOVA followed by Tukey’s Honest Significant difference test).

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Fig. 7. Levels of mRNA for osteogenic-specific genes (ALP, OCN, OPN, COLI, osteonectin, BSP and Rux2) of MG-63 cultured on NSF-PCL/2C, T2C, B2C and T/B 2C for 2 weeks. The mRNA levels were quantified using real-time RT-PCR and are normalized with respect to reference gene GAPDH. All genes were expressed significantly higher on T/B 2Cscaffolds *** p < 0.001, ** p < 0.01 and * p < 0.05, and data are presented as mean ± SD, n = 3.

(Fig. 1(c)). These may have contributed to improving viability and proliferation on NSF-PCL/2C, over NSF-PCL/3C. Composition and nomenclature of the growth factor loaded scaffolds are presented in Table 2. 3.4. Loading and release kinetics of growth factors The data for controlled release of the growth factors appear in Fig. 5(b) and suggest a burst release during the first 3 days for both growth factors. Overall though, only 19.33% of rhBMP-2 and 20.2% of TGF-␤ were released from NSF-PCL/2C scaffolds. The growth factors and HAp nuclei interacted to form a covalent bond–Scheme 3. This covalent linkage is likely to have slowed down growth factor release. 3.5. Cellular biocompatibility of growth factor loaded scaffolds 3.5.1. Cellular adhesion, viability, proliferation and total protein assessments MG-63 cells displayed better adhesion onto growth factor loaded scaffolds than the control (NSF-PCL/2C) (Fig. 6(a)). Between them, the growth factor loaded scaffolds did not display any significant difference in cell adhesion. During the initial hour, 66%, 87.11%, 87.98% and 91.53% of cells adhered onto NSF-PCL/2C, T2C, B2C and T/B2C scaffolds, rising to 89.22, 96.32, 96.95 and 98.81 over the following five hours. These results indicate that growth factor loading helped improve expression of fibronectin and specific integrin receptor subunits [45], leading to better cellular adhesion [46]. On day 1, cellular viability (Fig. 6 (b)) on the different scaffolds was not significantly different. For all scaffold compositions, cellular viability and proliferation (Fig. 6(c)) continually increased up to day 14. This increase may be taken to indicate lack of cytotoxicity among the nanofibrous scaffolds. Change of total protein content is presented in Fig. 6(d). The growth factor loaded scaffolds did not display a significant difference in their total protein content initially. Protein content on days 5, 9 and 14 was significantly higher than on day 1 for all scaffolds. By day 14, viability, cell proliferation and total protein content were significantly higher for T/B2C scaffolds than all the other scaffolds. 3.5.2. Gene expression by real-time RT-PCR mRNA expression of bone-associated genes was measured using RT-PCR to assess osteogenic potential of the fabricated scaffolds.

The results for the samples were averaged (n = 3) and the values normalized with respect to GAPDH, the housekeeping gene. Fig. 7 compares the gene expression from MG-63, upon the scaffolds, at day point 14. The T/B2 C scaffolds displayed significantly higher expression levels for genes evaluated. For bone sialoprotein (BSP), collagen I, osteopontin (OPN) and ALP, expressions were significantly better for B2C than T2C. No significant difference in expressions of osteocalcin (OCN), osteonectin and Runx2 was found between B2C and T2C. All growth factor loaded scaffolds gave significantly better levels of gene expression than NSF-PCL/2C.

3.5.3. Live/dead assay, cytoskeleton organization and cell morphology Results for the live/dead staining studies (Fig. 8), conducted at day point 5, showed most dense distribution of green coloured cells in the confocal images for T/B2C and B2C, followed by T2C and control. Cell cytoskeleton organization affects cell attachment and morphology. Images from examining the scaffolds under confocal microscope, after staining the nuclei and actin, are presented in Fig. 9. Z-scanning was used on the 3D scaffolds to examine the different layers. Scans for multiple layers were later merged into the final image presented. More extensive and uniform distribution of actin filaments could be observed for the growth factor loaded scaffolds, as compared to NSF-PCL/2C. Maximum numbers of cells were present on T/B2C, followed by T2C and B2C. For NSF-PCL/2C, cells were fewer and the actin distribution was sparse and limited to immediate vicinity of the cell nuclei. Cells on NSF-PCL/2C demonstrated round to oval forms and no characteristic orientation in the SEM images (Fig. 10(a) and (b)). Cells on T2C and B2C scaffolds had spindle-like structure, with extending filopodia/filament-like appendages. These scaffolds also had more extensive neo-matrix depositions compared to NSF-PCL/2C (Fig. 10(c)–(f)). Extensive deposits of cell-secreted neo-matrix on the T/B2C scaffolds obscured the underlying scaffold while most cells displayed near-flat morphology and good integration with each other, covering almost the entire surface (Fig. 10(g) and (h)). In these scaffolds, the nanofibrous structure and surface irregularities could improve initial cell adhesion [47]. The integrinbinding motifs of NSF, together with HAp, would be effective at improving the subsequent cellular processes.

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Fig. 8. The viability of MG-63 on different growth factors loaded nanofibrous scaffolds (NSF-PCL/2C, T2C, B2C and T/B 2C) using live/dead assay (live cells appear as green and dead cells as red) with confocal microscopy on day 5. High viability was maintained for T/B2C, followed by B2C and T2C. Magnification = 20×. Scale bar = 100 ␮m. (For interpretation of reference to color in this figure legend, the reader is referred to the web version of this article.)

3.5.4. ALP, ARS and calcium mineralization assay ALP is an established marker for osteogenic differentiation and the measured activity is presented (Fig. 11(b)), normalized with respect to the cell density. Cell density was obtained using DNA analysis after 2 weeks of culture (Fig. 11(a)). Average cell density on T/B2C scaffold was 3.78 × 104 cells/scaffold. For T2C and B2C, the numbers were 3.39 × 104 and 3.51 × 104 , respectively. Thus, compared to day 1’s attached cells, cell numbers rose by 93.16% (T/B 2C), 81.56% (B2C) and 76.32% (T2C). For the NSF-PCL/2C scaffold, cell density increase was only 65%. Maximum ALP activity resulted in the T/B2C scaffolds, followed by B2C and T2C. These findings confirm that scaffolds with growth factor incorporation can enhance differentiation of the seeded osteogenic cells. As seen from Alizarin Red-S (ARS) staining results, all composite scaffolds had developed mineral deposits at day 9 (Fig. 11(c)). These

deposits grew by day 14. Calcium deposition assay, used to further confirm mineralization (Fig. 11 (d)), showed similar trends as the ARS staining (Fig. 11 (c)). Calcium deposits on all scaffolds consistently increased during these two weeks. Both analyses showed that calcium deposits were significantly more (p < 0.01) on T/B2C scaffold. These results had a trend similar to that of cell proliferation. However, B2C gave significantly more calcium content (p < 0.05) than T2C. Greater mineral deposits in the T/B2C scaffolds indicate greater degree of differentiation among the cells on those scaffolds, thus implying enhanced potential for bone regeneration. Presence of ECM is a requirement for successful tissue reconstruction and formation of ordered ECM relies on effective adherence of cells onto scaffolds [48]. A multitude of factors, as the following, conceivably contribute to the more favourable cytocompatibility of T/B2C: nano-scaled structure (in terms of fibre

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Fig. 9. The cytoskeletal actin organization and distribution of MG-63 cells grown on different growth factors loaded nanofibrous scaffolds (NSF-PCL/2C, T2C, B2C and T/B 2C) at day point 7. The confocal images were taken after staining the actin filaments with Alexa Fluor® 488 (green) and counterstaining with Hoechst 33342 (blue) for nuclei. Better actin organization, cell–cell contiguity and larger cell numbers were observed on T/B2C, compared to T2C and B2C. Magnification = 20×. Scale bar = 100 ␮m. (For interpretation of reference to color in this figure legend, the reader is referred to the web version of this article.)

diameter), inherent integrin-binding peptide (RGD) sequences in this particular NSF, favourability of calcium phosphate for proliferation of osteoblast-like cells and dual growth factors delivery. Progression of cell proliferation from day 5 to day 9 is a little slow for all the scaffolds (Fig. 6 (c)). This slightly restricted progress may be ascribed to the cells requiring some time to adjust and adapt to the 3D matrix, on being transferred from the 2D cultures [49]. Previous works have demonstrated successful bone regeneration, using single growth factors incorporated into polymeric

substrates [50,51], but with growth factor concentrations at supraphysiological levels. Physiological level for BMP is ∼1 ␮g/gm bone tissue [52]. Economic impracticality of using immensely higher amounts of growth factors can render a technique infeasible for regular clinical usage [22]. The current work uses growth factors at near-physiological concentrations (approximately 4–104 ng protein/g scaffold), in addition to dual growth factor delivery. rhBMP-2 and TGF-␤ were chosen for the combined delivery since both these growth factors have independent roles during natural regeneration of bone. The in vitro results show significant advantages of dual growth factor delivery, thus validating the initial hypothesis

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Fig. 10. Scanning electron micrographs of the osteoblast-like cells (MG-63) seeded on the different growth factors loaded nanofibrous scaffolds (NSF-PCL/2C, T2C, B2C and T/B 2C) after 14 days of culture. The cells formed continuous multi-layer sheets on (g, h) dual (T/B 2C) and (c–f) single growth factor loaded scaffolds (T2C and B2C), with large amount of neo-matrix deposition, compared to a few isolated cells on the (a, b) without growth factor loaded (NSF-PCL/2C) matrix. Scale bar = 10 ␮m. Magnification = 1K× (a, c, e, g) and 3.5K× (b, d, f, h).

put forth regarding effect of using dual growth factor. Optimizing growth factor delivery mode, rate, quantity and proportion could be part of further investigations to bring about more improvements in these fabricated scaffolds. Polymeric scaffolds with multiple such growth factors incorporated, that have their distinct kinetics [53,54], can provide a starting point for mimicking the growth factor expression sequence during natural bone regeneration [18].

Overall, this work showed that mineralizing NSF grafted PCL nanofibrous matrices with HAp improved their biocompatibility, mechanical strength, cell viability and proliferation. Subsequently, incorporation of dual growth factors (rh BMP-2 + TGF-␤) on the NSF-PCL/2C scaffold was a noticeably better choice as regards biocompatibility and adhesion, proliferation and differentiation of cells.

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Fig. 11. (a) Cell count through DNA analysis, (b) alkaline phosphatase (ALP) activity, (c) quantification of mineral deposition using ARS staining and (d) calcium deposition of osteoblast-like cells (MG-63) seeded on different growth factors loaded nanofibrous scaffolds (NSF-PCL/2C, T2C, B2C and T/B2C) after 14 days of cell culture. ALP activity was reported as p-nitrophenol produced, normalized by incubation duration and cell count of ␮mol/min/104 cells. ALP activity of all the constructs increased with time, the highest activity observed on T/B2C. Both mineral deposition (Ca2+ ) studies (c, d) provided same trend and highest calcium deposition was observed for T/B2C. *** p < 0.001; ** p < 0.01; * p < 0.05, n = 3 at each time point (One-way ANOVA followed by Tukey’s Honest significant difference test).

4. Conclusion

Funding

The alternate soaking procedure provided almost uniform HAp deposition upon fibroin grafted PCL nanofibrous matrices. HAp deposition quantity had a direct correlation with the number of cycles of soaking used. Introduction of HAp improved the scaffolds’ hydrophilicity. The HAp content obtained from 2 cycles of coating provided best strength and biocompatibility. Detailed in vitro studies were conducted after growth factor loading on the 2 cycle HAp coated scaffolds. Findings of these detailed studies indicate the better performance of combined growth factor loaded scaffolds at supporting cell adhesion and proliferation, ECM formation and expression of bone-associated genes. These findings validate the starting hypothesis. These dual growth factor loaded scaffolds show definite promise in terms of a suitable construct for bone tissue engineering. The scaffold material examined in this work could lead to improved man-made biocomposites targeting bone reconstructions. On-going investigations by our group are examining aspects of the scaffold design and growth factor loading for an optimal solution. This would culminate in in vivo testing and comparative studies on the effect of mineralization methodology.

Department of Biotechnology (BT/PR10941/MED/32/333/2014) and Indian Council of Medical Research (5/13/12/2010/NCD-III), Govt. of India. Acknowledgements Department of Biotechnology and Indian Council of Medical Research, Govt. of India supported the work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.apmt.2016.09.007. References [1] S. Mann, Molecular recognition in biomineralization, Nature 332 (6160) (1988) 119–124. [2] C. Cheng, Y. Yang, X. Chen, Z. Shao, Templating effect of silk fibers in the oriented deposition of aragonite, Chem. Commun. (43) (2008) 5511–5513.

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